Methanocarba cycloakyl nucleoside analogues

ABSTRACT

The present invention provides novel nucleoside and nucleotide derivatives that are useful agonists or antagonists of P1 or P2 receptors. For example, the present invention provides a compound of formula A-M, wherein A is modified adenine or uracil and M is a constrained cycloalkyl group. The adenine or uracil is bonded to the constrained cycloakyl group. The compounds of the present invention are useful in the treatment or prevention of various diseases including airway diseases (through A 2B , A 3 , P2Y 2  receptors), cancer (through A 3 , P2 receptors), cardiac arrhythmias (through A 1  receptors), cardiac ischemia (through A 1 , A 3  receptors), epilepsy (through A 1 , P2X receptors), and Huntington&#39;s Disease (through A 2A  receptors).

CROSS-REFERENCE TO A RELATED APPLICATION

[0001] This application claims the benefit of U.S. provisionalapplication No. 60/176,373, filed Jan. 14, 2000, the disclosure of whichis incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

[0002] This invention pertains to a novel class of receptor ligands forP1 and P2 receptors and their therapeutic use. More specifically, theinvention pertains to nucleoside derivatives in which the sugar moietyis replaced with a cycloalkyl group that is conformationally constrainedby fusion to a second cycloalkyl group.

BACKGROUND OF THE INVENTION

[0003] Purines such as adenosine have been shown to play a wide array ofroles in biological systems. For example, physiological roles played byadenosine include, inter alia, modulator of vasodilation andhypotension, muscle relaxant, central depressant, inhibitor of plateletaggregation, regulator of energy supply/demand, responder to oxygenavailability, neurotransmitter, and neuromodulator. (Bruns, Nucleosides& Nucleotides, 10(5), 931-934 (1991)). Because of its potent actions onmany organs and systems, adenosine and its receptors have been thesubject of considerable drug-development research (Daly, J. Med. Chem.,25, 197 (1982)). Potential therapeutic applications for agonistsinclude, for instance, the prevention of reperfusion injury aftercardiac ischemia or stroke, and treatment of hypertension and epilepsy(Jacobson, et al., J. Med. Chem., 35, 407-422 (1992)). Adenosine itselfhas recently been approved for the treatment of paroxysmal supraventricular tachycardia (Pantely, et al., Circulation, 82, 1854 (1990)).Adenosine receptor agonists also find use as anti-arrhythmics,antinociceptives, anti-lipolytics, cerebroprotectives, andantipsychotics.

[0004] P2 receptors, are present in heart, skeletal, various smoothmuscles, prostate, ovary, and brain and have been implicated in certainaggregation processes associated with thrombosis and asanti-hypertensive and anti-diabetic agents. Agonists that bind the P2receptor induce activation of phospholipase C, which leads to thegeneration of inositol phosphates and diacyl glycerol with a subsequentrise in intracellular calcium concentration and muscle relaxation. P2receptor antagonists block ADP-promoted aggregation in platelets andthereby exert an anti-thrombotic effect.

[0005] All P1 and P2 receptor nucleoside ligands suffer from chemicalinstability that is caused by the labile glycosidic linkage in the sugarmoiety of the nucleoside. However, it has been found that relatively fewribose modifications are tolerated by the presently known agonists andantagonists of P1 and P2 receptors.

[0006] New compositions are needed that have improved chemical stabilityand that do not destroy the activity of such compounds.

[0007] The invention provides such compositions and methods of usingthem in the treatment of disease. These and other advantages of thepresent invention, as well as additional inventive features, will beapparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

[0008] The present invention provides novel nucleoside and nucleotidederivatives that are useful agonists or antagonists of P1 or P2receptors. The invention is premised upon the novel combination ofadenine and uracil and their derivatives with a constrained cycloalkylgroup, typically a cyclopentyl group. The constraint on the cycloalkylgroup is introduced by fusion to a second cycloalkyl group. In the caseof cyclopentane, the fusion is typically with cyclopropane. The presentcompounds retain a surprising binding affinity despite the substitutionfor the ribose group. Moreover, the absence of the glycosidic bond inthe compounds assists in improving the chemical stability of thecompounds and aids in overcoming the stabilit problem associated withthe glycosidic bond in previously known P1 and P2 receptor ligands.

[0009] The compounds of the present invention are useful in thetreatment or prevention of various airway diseases (through A_(2B), A₃,P2Y₂ receptors), cancer (through A₃, P2 receptors), cardiac arrhythmias(through A₁, receptors), cardiac ischemia (through A₂, A₃ receptors),epilepsy (through A₁, P2X receptors), Huntington's Disease (throughA_(2A) receptors), Immunodeficient disorders (through A₂, A₃ receptors),inflammatory disorders (through A₃, P2 receptors), neonatal hypoxia(through A₁ receptors), neurodegenerative (through A₁, A₃, P2receptors), pain (through A₁, A₃, P2X3 receptors), Parkinson's Disease(through A_(2A) receptors), renal failure (through A₁ receptors),schizophrenia (through A_(2A) receptors), sleep disorders (through A₁receptors), stroke (through A₂, A₃, P2 receptors) thrombosis (throughP2Y₁, P2Y_(AC) receptors), urinary incontinence (through P2X₁,receptors), diabetes (through A₁, receptors), psoriasis (through P2Xreceptors), septic shock (through P2 receptors), brain trauma (throughA₁, receptors), glaucoma (through A₃ receptors) and congestive heartfailure (through P2 receptors).

[0010] The invention may best be understood with reference to theaccompanying drawings and in the following detailed description of thepreferred embodiments.

DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention provides a new class of nucleoside andnucleotide analogs that serve as selective agonists or antagonists forP1 and P2 receptors.

[0012] Generally, the compounds of the present invention comprise twobasic chemical components designated “A” and “M” which are covalentlybonded to one another. Component A comprises adenine or uracil, andcomponent M includes a constrained cycloalkyl group. Preferably theadenine and uracil are chemically modified or substituted with moietiesthat allow the compound to bind to a P1 or P2 receptor. To that end anyof a wide variety. of chemical groups can be used to modify adenine anduracil. Those groups are well known to those of skill in the receptorart. Preferably, when A is purine or a purine derivative, the linkagebetween A and M is a chemical bond between the N9 purine nitrogen andthe C1 carbon of the cycloalkyl group. Where A is pyrimidine or apyrimidine derivative, the bond is between N1 pyrimidine nitrogen andthe C1 carbon of the cycloalkyl group. The compounds of the presentinvention have improved stability and surprising receptor bindingaffinity.

[0013] While not wishing to be bound to any particular theory, it isbelieved that the constrained cycloalkyl group assists in improvingchemical stability and receptor affinity. Preferably the cycloalkylgroups are capable of adopting a conformation such that the compound canbind to P1 or P2 receptors. As a result, preferred cycloalkyl groups arethose that tend to form energetically favorable interactions with P1 andP2 receptors and avoid energetically unfavorable ones, such asunfavorable ionic and/or steric interactions. Further, the cycloalkylgroup is derivatized with a bridging group. The constraint restricts thecycloalkyl group to certain conformations that are believed to bebeneficial to binding affinity. The preferred cycloalkyl group is acyclopentyl group. With cyclopentyl groups the preferred method forintroducing a conformational constraint is by derivatizing with a fusedcyclopropane bridge. With this modification the cyclopentane ring isbelieved to be constrained to mimic the conformation of a rigid furanosering.

[0014] Compounds of the present invention include the compounds shownbelow in Formulae I and II.

[0015] Formulae I and II show compounds in which a derivatized orunderivatized adenine base is joined to a constrained cyclopentyl group.For purposes of reference, the carbon atom of the cyclopentyl group, M,that is joined to adenine, A, is the C1 carbon and the adenine is joinedto M through its N9 nitrogen. In the compounds of Formulae I and II theconstrained cyclopentyl group is derivatized with a fused cyclopropanebridge. In Formula I the cyclopropyl group bridges carbon atoms C4 andC6. In Formula II the cyclopropyl group bridges carbon atoms C6 and C1.These distinct bridging patterns constrain the cyclopentyl group intodistinct conformations, specifically the N-(northern) conformation as inFormula I and the S-(southern) conformation as in Formula II. These twoconformations are thought to mimic the two biologically activeconformations of furanose groups for P1 and P2 receptor binding pockets.

[0016] The compounds described by Formulae I and II can be furtherdefined by a variety of suitable modifications to the adenine group. Asdiscussed above, any of a wide variety of chemical groups can be used toform suitable adenine derivatives that comprise the novel compounds ofthe present invention, provided that the resulting compound is capableof binding to a P1 or P2 receptor. These chemical groups are well knownin the art and have been described, for example in U.S. Pat. Nos.5,284,834; 5,498,605; 5,620,676; 5,688,774; and Jacobson and Van Rhee,PURINERGIC APPROACHES IN EXPERIMENTAL THERAPEUTICS, Chapter 6, p. 101(Jacobson and Jarvis eds., 1997); and Jacobson et al., The P2 NucleotideReceptors, P. 81-107, in The Receptors (Turner et al. eds. 1998), whichare incorporated by reference herein. The combination of the chemicallymodified adenine and the constrained cycloalkyl group provides asurprising improvement in both chemical stability and binding affinity.

[0017] By way of example and not in limitation of the present inventionin the compounds of Formulae I and II, R₁ is hydrogen, alkyl,cycloalkyl, alkoxy, cycloalkoxy, aryl, arylalkyl, acyl, sulfonyl,arylsulfonyl, thiazolyl or bicyclic alkyl; R₂ is hydrogen, halo, alkyl,aryl, arylamino, aryloxide, alkynyl, alkenyl, thioether, cyano,alkylthio or arylalkylthio; R₃, R₄, and R₅, are each hydrogen, hydroxyl,alkoxy, alkyl, alkenyl, alkynyl, aryl, acyl, alkylamino, arylamino,phosphoryl, phosphonyl, boronyl, or vanadyl, and they can be the same ordifferent; R₆ is hydrogen, alkyl, alkenyl, alkynyl, or aminoalkyl. R₇ isa methylene, dihalomethyl, carbonyl, or sulfoxide group. R₈ is carbon ornitrogen. At least one of R₁, R₂, and R₆ is not hydrogen. It can beappreciated that various combinations of the above groups are alsowithin the invention provided that they retain agonist or antagonistactivity with a P1 or P2 type receptor.

[0018] Where an alkyl, alkenyl, alkynyl group is referenced by itself oras part of another group, the reference is to an uninterrupted carbonchain consisting of no more than 20 carbon atoms. Aryl and cycloalkylgroups contain no more than 8 carbons in the ring.

[0019] Reference to alkyl groups is further meant to include straight orbranched chain alkyls, arylalkyl, aminoalkyl, haloalkyl, alkylthio orarylalkylthio groups. Alkyls specifically include methyl throughdodecyl. Where alkyl groups are present at position R₆ in adenine, it ispreferred that the chain length be no longer than 6 carbons. Arylalkylgroups include, phenylisopropyl, phenylethyl. Aminoalkyl groups can beany suitable alkyl group also containing an amine. Similarly, haloalkylgroups can be any suitable alkyl group that contains a halo substituent,such as bromo, chloro, flouro, iodo. Alkylthio includes such moieties asthiomethyl, thiopentyl, thiohexyl, thioheptyl, thiooctyl, thiodecyl,thioundecyl, ethylthioethyl, or 6-cyanohexylthio groups. Alkylthio alsois meant to include arylalkylthio such as 2-(p-nitrophenyl)ethyl)thio,2-aminophenylethylthio, 2-(p-nitrophenyl)ethylthio, or2-aminophenylethylthio.

[0020] Cycloalkyls for example cyclopentyl, cyclohexyl,hydroxycyclopentyl.

[0021] Alkoxys include for example methoxy groups.

[0022] Cycloalkoxys can include cyclopentoxy.

[0023] Aryl moieties can be arylalkyl, arylalkylthio, arylsulfonyl,arylamino, aryloxide, heteroaryl, haloaryl, arylurea, arylcarboxamido,heteroarylamino or sulfoaryl. Benzyl groups are one species of arylgroup. In addition, the arylalkyls include R-phenylisopropyl orphenylethyl. Aryloxides can be phenyl, R-phenylisopropyl, phenylethyl,3,5-dimethoxyphenyl-2-(2-methylphenyl)ethyl and sulfophenyl. Haloarylcan be iodobenzyl among other halogenated aryl groups. Additionally, theheteroaryls include, for example, furans such as tetrahydrofuran.

[0024] Acyl groups include carbonyls.

[0025] Alkenyl groups are analogous to alkyl groups but include at leastone carbon-carbon double bond. When present at the R₆ group of adenineit is preferred that the carbon chain length be from 2 to 6 carbons.

[0026] Similarly, alkynyls are analogous to alkenyl groups but containat least one triple carbon-carbon bond. As with other groups, whenpresent at the R₆ position of adenine it is preferred that they are notlonger than 6 carbons.

[0027] Phosphoryl groups include diphosphoryl, triphosphoryl,thiophosphoryl, thiodiphosphoryl, thiotriphosphoryl, imidodiphosphate,imidotriphosphate, methylene diphosphate, methylenetriphosphate,halomethylene diphosphate, halomethylene triphosphate, boranophosphate,boranodiphosphate, boranotriphosphate, or phosphorothioate-2-thioetherfor example.

[0028] Thio groups include alkylthio, arylalkylthio, alkenylthio, orarylthios. Alkylthio includes such groups as thiomethyl, thiopentyl,thiohexyl, thioheptyl, thiooctyl, thiodecyl, thioundecyl,ethylthioethyl, or 6-cyanohexylthio. Alkenylthio includes 5-hexenylthio.Arylthios include 2-(p-nitrophenyl) ethyl)thio, 2-aminophenylethylthio,2-(p-nitrophenyl) ethylthio, or 2-aminophenylethylthio.

[0029] One example of a suitable. thiazolyl is(benzothiazolyl)thio-2-propyl.

[0030] Examples of bicycloalkyls include s-endonorbornyl, orcarbamethylcyclopentane.

[0031] Halo groups include such elements as fluoro, bromo, chloro, oriodo.

[0032] It will also be appreciated that any group that may be furthersubstituted can be, and still be within the scope of the invention. Forexample, all of the R₁ groups except hydrogen can be furthersubstituted. By way of illustration, when R₁ is not hydrogen, it can befurther modified by substitutions with any of the following chemicalsubstituents including amino, cyano, alkoxyl, alkyl, alkenyl, alkynyl,cycloalkyl, aryl, arylalkyl, acyl, halo, hydroxy, phosphoryl, sulfonyl,sulfonamido, carboxyl, thiohydroxyl, sulfonamido, carboxyl, andcarboxamido groups. Similarly, for R₂-R₁₀ all of the groups other thanhydrogen can be substituted further. Multiple substitutions are alsocontemplated.

[0033] In a preferred embodiment R₁ can be either methyl, cyclopentyl,cyclohexyl, phenyl, R-phenylisopropyl, benzyl, or phenylethyl; R₂ ischloride; and R₆ can be a C₁-C₆ alkylamino, C₁-C₆ alkyl, C₁-C₆ alkenyl,C₁-C₆ alkynyl group.

[0034] Other compounds of the present invention include the compoundsshown below in Formulae III and IV. The Formulae show compounds in whicha derivatized or underivatized uracil base is joined to a constrainedcyclopentyl group.

[0035] The compounds defined by formulae III and IV can be furtherdefined by a variety of suitable modifications. For example R₁ can behydrogen, or an alkyl group; R₂ can be hydrogen, C₁-C₆ alkyl, C₁-C₆alkenyl, C₁-C₆ alkynyl, or a C₁-C₆ aminoalkyl group; R₃, R₄, R₅, caneach independently be the same as discussed previously with respect toFormulae I and Formulae II. R₆ and R₇ are each independently eithersulfur or oxygen.

[0036] Certain compounds of the present invention are ligands of P2receptors. A variety of P2 receptors are known in the art and thepresent compounds act at one or more of these, which include forexample, P2X and P2Y receptors. These Receptor ligands are compoundsthat bind receptors, preferably in the binding pocket. In certainembodiments the compound can be a P2 receptor agonist. In otherembodiments the compound can be a P2 receptor antagonist.

[0037] Certain compounds of the present invention are ligands for the P1receptor. A variety of subclasses of P1 receptors are known and variousof present compounds act at one or more these species, which include forexample A₁, A₂, and A₃ receptors. Certain compounds act as P1 receptoragonists while others appear to act as antagonists.

[0038] The compounds of the present invention are useful in thetreatment or prevention of various airway diseases (through A_(2B), A₃,P2Y₂ receptors), cancer (through A₃, P2 receptors), cardiac arrhythmias(through A₁ receptors), cardiac ischemia (through A₁, A₃ receptors),epilepsy (through A₁, P2X receptors), Huntington's Disease (throughA_(2A) receptors), Immunodeficient disorders (through A₂, A₃ receptors),inflammatory disorders (through A₃, P₂ receptors), neonatal hypoxia(through A₁ receptors), neurodegenerative (through A₁, A₃, P2receptors), pain (through A₁, A₃, P2X3 receptors), Parkinson's Disease(through A_(2A) receptors), renal failure (through A₁ receptors),schizophrenia (through A_(2A) receptors), sleep disorders (through A₁receptors), stroke (through A₁, A₃, P2 receptors), thrombosis (throughP2Y₁, P2Y_(AC) receptors), urinary incontinence (through P2X₁receptors), diabetes (through A₁ receptors), psoriasis (through -P2Xreceptors), septic shock (through P2 receptors), brain trauma (throughA₁ receptors), glaucoma (through A₃ receptors), and congestive heartfailure (through P2 receptors).

[0039] The present invention is further directed to a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and atleast one compound selected from the group consisting of the presentlydescribed compounds.

[0040] The pharmaceutically acceptable excipients described herein, forexample, vehicles, adjuvants, carriers or diluents, are well-known tothose who are skilled in the art and are readily available to thepublic. It is preferred that the pharmaceutically acceptable carrier beone that is chemically inert to the active compounds and one that has nodetrimental side effects or toxicity under the conditions of use.

[0041] The choice of excipient will be determined in part by theparticular compound of the present invention chosen, as well as by theparticular method used to administer the composition. Accordingly, thereis a wide variety of suitable formulations of the pharmaceuticalcomposition of the present invention. The following formulations fororal, aerosol, parenteral, subcutaneous, intravenous, intramuscular,interperitoneal, rectal, and vaginal administration are merely exemplaryand are in no way limiting.

[0042] One skilled in the art will appreciate that suitable methods ofutilizing a compound and administering it to a mammal for the treatmentof disease states, which would be useful in the method of the presentinvention, are available. Although more than one route can be used toadminister a particular compound, a particular route can provide a moreimmediate and more effective reaction than another route. Accordingly,the described methods are merely exemplary and are in no way limiting.

[0043] The dose administered to an animal, particularly human and othermammals, in accordance with the present invention should be sufficientto effect the desired response. Such responses include reversal orprevention of the bad effects of the disease for which treatment isdesired or to elicit the desired benefit One skilled in the art willrecognize that dosage will depend upon a variety of factors, includingthe age, species, condition or disease state, and body weight of theanimal, as well as the source and extent of the disease condition in theanimal. The size of the dose will also be determined by the route,timing and frequency of administration as well as the existence, nature,and extent of any adverse side-effects that might accompany theadministration of a particular compound and the desired physiologicaleffect. It will be appreciated by one of skill in the art that variousconditions or disease states may require prolonged treatment involvingmultiple administrations.

[0044] Suitable doses and dosage regimens can be determined byconventional range-finding techniques known to those of ordinary skillin the art. Generally, treatment is initiated with smaller dosages thatare less than the optimum dose of the compound. Thereafter, the dosageis increased by small increments until the optimum effect under thecircumstances is reached. The present inventive method typically willinvolve the administration of about 0.1 to about 300 mg of one or moreof the compounds described above per kg body weight of the individual.

[0045] The following examples further illustrate the present inventionbut, of course, should not be construed as in any way limiting itsscope. In the examples, unless otherwise noted, compounds werecharacterized and resonances assigned by 300 MHz proton nuclear magneticresonance mass spectroscopy using a Varian GEMINI-300 FT-NMRspectrometer. Also, unless noted otherwise, chemical shifts areexpressed as ppm downfield from tetramethylsilane. Syntheticintermediates were characterized by chemical ionization massspectrometry (NH₃) and adenosine derivatives by fast atom bombardmentmass spectrometry (positive ions in a noba or m-bullet matrix) on a JEOLSX102 mass spectrometer. Low resolution CI-NH₃ (chemical ionization)mass spectra were carried out with Finnigan 4600 mass spectrometer andhigh-resolution EI (electron impact) mass spectrometry with a VG7070Fmass spectrometry at 6 kV. Elemental analysis was performed by AtlanticMicrolab Inc. (Norcross, Ga.). NMR and mass spectra were consistent withthe assigned structure.

EXAMPLE 1

[0046] In all of the potent adenosine agonists previously developed, theribose moiety is present, and consequently, these agonists are subjectto deglycosylation and other pathways of metabolic degradation in vivo;In order to design non-glycosyl adenosine agonists and thereby increasebiological stability and potential receptor selectivity, carbocyclicmodifications of the ribose moiety have been introduced. In previousstudies of adenosine analogues it was found that if adenosinederivatives having carbocyclic modifications of the ribose ring(compounds 1-4, below) bind to adenosine receptors it is only withgreatly reduced affinity.

[0047] In the present study we have incorporated a complex carbocyclicmodification of ribose for use with adenosine agonists. Thismodification, wherein only one isomeric form retains high affinity andreceptor selectivity, is the “methanocarba” ring. In this modification afused cyclopropane ring constrains the accompanying cyclopentane moietyto mimic the conformation of a rigid furanose ring. The furanose ring ofnucleosides and nucleotides in solution is known to exist in a rapid,dynamic equilibrium between a range of Northern and opposing Southernconformations as defined in the pseudorotational cycle. For methanocarbaanalogues, the bicyclo[3.1.0]hexane ring can constrain the cyclopentanering into a N-, 2′-exo envelope pucker, and a S-, 3′ exo form.

[0048] These two extreme forms of ring pucker usually definebiologically active conformations. This example shows that nucleosidebinding to P1-(adenosine) receptors, is favored when the fixedring-twist conformation is in the N-conformation.

[0049] Chemical Synthesis.

[0050] Nucleosides and synthetic reagents were purchased from SigmaChemical Co. (St. Louis, Mo.) and Aldrich (St. Louis, Mo.).2,6-Dichloropurine was obtained from Sigma. m-iodobenzyl bromide waspurchased from Aldrich (St. Louis, Mo.).4-(6-Aminopurin-9-yl)-1-hydroxymethyl-bicyclo[3.1.0]hexane-2,3-diol (1)and compounds 5c and 5d were obtained from Dr. Victor Marquez. Compounds7a and 9a were synthesized in our laboratory.

[0051] The synthetic strategy used in this example is shown below. Thesynthesis of N6-substituted N-methanocarba adenosine derivativesoptimized for interaction with Al (CP=cyclopentyl) or A3(IB=3-iodobenzyl) receptors. Reagents: a) DEAD, Ph₃P; b) MEOH, rt; c)BC1₃; d) H2/Pd; e) 3-iodobenzyl bromide, 50° C., DMF, 2 days; f) NH₄0H,MEOH, 80° C., 3 days.

[0052] (1′R, 2R, 3′R, 4′R,1′aR)-2,3-(dihydroxy)-4-(hydroxymethyl)-1-(6-cyclopentylaminopurine-9-yl)bicyclo(3.1.0)hexane)(6c):

[0053] A solution of 8c (4 mg, 0.01 mmol) in methanol (0.5 ml) washydrogenated at atmospheric pressure over 10% Pd/C (1 mg) to furnish theproduct 6c (83% yield). H¹NMR (CD₃0D): δ0.7-0.8 (m, IH, 6′-CHH),1.46-1.88 (m, 1OH, 6′CHH, 1′aH, 4CH₂), 2.01-2.20 (m, 1H, NCH), 3.34 (d,1H, J=9.77 Hz, 5′CHH), 3.88 (d, 1H, J=6.84 Hz, 3′CH), 4.26 (d, 1H,J=9.77 Hz, 5′CHH), 4.66-498 (m, 2H, 2′CH, 1′CH), 8.28 (s, 1H, 2CH), 8.5(s, 1H, 8CH). HRMS(FAB): Cal: 346.1879 Found: 346.1879

[0054] (1′R, 2′R, 3′R,4′R)-2,3-(dihydroxy)-4-(hydroxymethyl)-1-(6-(3-idobenzylamino)purine-9-yl)cyclopentane(7b):

[0055] A mixture of aristeromycin (3.5 mg, 0.013 mmol) and3-iodobenzybromide (12 mg, 0.039 mmol) in anhydrous DMF was heated for 3days, and solvent was removed under vacuum. The excess 3-iodobenzylaminewas removed from the reaction mixture by adding ether to the reactionmixture, and stirring was continued for 5 min. followed by decantationof the supernatant ether phase. The residue was dried, suspended inmethanol (1 ml) and ammonium hydroxide (0.5 ml), and heated at 80° C. ina closed tube for 1 h. Solvent was removed under vacuum, and the residueobtained was purified by flash column chromatography using 7/3chloroform/methanol to furnish 3.0 mg (47%) of the product.

[0056] H¹NMR(CD₃0D) δ1.86-1.96 (m, 1H, 1′CHH), 2.14-2.30 (m, 1H, 1′CHH),2.38-2.48 (m, 1H, 4′CH), 3.3-3.38 (m, 1H, 5′CHH), 3.67 (d, 1H, J=6.84Hz, 5′CHH), 3.96-4.06 (m, 1H, 3′CH), 4.43-4.48 (m, 1H, 2′CH), 4.73-4.82(m, 1H, 1′CH), 5.26 (s, 2H, ArCH₂), 7.12 (t, 1H, J=7.82 Hz, ArH), 7.32(d, 1H, J=7.82 Hz, ArH), 7.66 (d, 1H, J=7.82 Hz, ArH), 7.73 (s, 1H,ArH), 8.06 (s, 1H, 2CH). 8.08 (s, 1H, 8CH).

[0057] Preparation of 4-[6-(3-iodobenzylamino)-purin-9-yl]-l-hydroxymethyl-bicyclo[3.1.0]hexane-2,3-diol (7c,(N)-Methanocarba-N⁶-(3-iodobenzyl)adenosine) by Dimroth rarrangement:¹

[0058] To a solution of 4-(6-amino-purin-9-yl)-1-hydroxymethyl-bicyclo[3.1.0]hexane-2,3-diol (5c, 20 mg, 0.0721 mmol)in DMF (0.5 mL) was added m-iodobenzyl bromide (64 mg, 0.216 mmol), andthe mixture was stirred at 50° C. for 2 days. DMF was then removed undera stream of N₂. To the resulting syrup 0.5 mL of acetone and 1 mL ofether were added and the syrup solidified. The solvents were removed bydecantation, and again ether was added and removed. The solid was driedand dissolved in 1 mL MEOH. NH₄OH (1.5 mL) was added and the mixture wasstirred at 80° C. for 3 days. After cooling down to room temperature,the solvents were removed under reduced pressure and the residue waspurified by preparative TLC (silica 60; 1 000 μm; Analtech, Newark,Del.; ethyl acetate-i-PrOH-H₂O (8:2:1)) to give 26 mg of the product(7c), yield: 73%. ¹ H NMR (CDC1₃): δ0.82 (t, J=6.0 Hz, 1 H), 1.41 (t,J=4.8 Hz, 1 H), 1. 72 (dd, J=8.5, 6. 0 Hz, 1H), 3.36 (d, J=10.8 Hz, 1H), 4.05 (d, J=6.9 Hz, 1 H), 4.33 (m, 1 H), 4.80-4.88 (m, 3 H), 5.21 (d,J=6.9 Hz, 1 H), 6.25 (m, br, 1), 7.07 (t, J=7.8 Hz, 1 H), 7.35 (d, J=7.8Hz, 1 H), 7.61 (d, J=7.8 Hz, 1 H), 7.74 (s, 1), 7.93 (s, 1 H), 8.33 (s,1 H). MS(FAB): m/z 494 (M³⁰+I). (1′R, 2′R, 3′R, 4′R,l′aR,)-2,3-(dihydroxy)-4-(hydroxymethyl)-1-(2-chloro-6-cyclopentylaminopurine-9-yl)bicyclo(3.1.0)hexane)(8c):

[0059] To a solution of 15 (36 mg, 0.076 mmol) in anhydrousdichloromethane was added BCl₃ (1M solution in dichloromethane, 0.23 ml,0.23 mmol) at 0° C. The reaction mixture was warmed to room temperatureand stirred for 10 min. To this mixture was added methanol (1 ml)followed by ammonium hydroxide (0.5 ml). The mixture was concentratedunder vacuum, and the residue obtained was purified by flash columnchromatography using 9/1 chloroform-1/methanol as eluent to furnish 14mg of the product 8c (48% yield) as a solid.

[0060] H¹NMR(CDCl₃): δo.65-0.9 (m, IH, 6′CHH), 1.1-1.4 (m, 2H, 6′CHH,1′aH), 1.4-1.9 (m, 8H, 4CH₂), 2.0-2.2 (m, 1H, N⁶CH), 3.34 (d, 1H, J=7.2Hz, 5′CHH), 3.97 (d, 1H, J=4.6 Hz, 3′CH), 4.25 (d, 1H, J=7.2 Hz, 5′CHH),4.687 (s, 1H, 1′CH), 5.11 (d, 1H, J 4.6, 240 CH), 7.85 (s, 1H, 8CH).HRMS(FAB): Cal: 380.1489 found: 380.1498

[0061] (1′R, 2′R, 3′R, 4′R,l′aR)-2,3-(dihydroxy)-4-(hydroxymethyl)-1-(2-chloro-6-(3-idobenzylamino)purine-9-yl)bicyclo(3.1.0)hexane)(9c) was synthesized by the same method as 8c in 53% yield.

[0062] H¹NMR(CD₃OD): δ0.70-0.78 (m, 1H, 6′CHH), 1.50-1.63 (m, 2H, 6,,CHH, 1′aH), 3.33 (d, 1H, J=11.72 Hz, 5′CHH), 3.88 (d, 1H, J=6.84 Hz, 340CH), 4.26 (d, 1H, J=11.72 Hz, 5′CHH), 4.71-4.83 (m, 2H, 1′CH, 2′CH), 7.1(t, 1H, J=7.82 Hz, ArH), 7.40 (d, 1H, J=7.82 Hz, ArH), 7.61 (d, 1H, 7.82Hz, ArH), 7.78 (s, 1H, ArH), 8.54 (s, 1H, 8CH). HRMS(FAB): Cal: 528.0299Found: 528.0295

[0063] (2R, 3R, 4R, l′aR,1S)-2,3-(O-isopropylidine)-4-(methylenebenzyloxy)-1-(2,6dichloropurine-9-yl)bicyclo(3.1.0)hexane)(12):

[0064] To a solution of triphenyl phosphine (260 mg, 1 mmol) inanhydrous THF (2 ml) was added DEAD (0.16 ml, 1 mmol) dropwise at 0° C.,and stirring was continued for 20 min. To this solution was added asolution of 2,6-dichloropurine in THF (4 ml) followed by the addition of11 (145 mg, 0.5 mmol) in THF (4 ml). The reaction mixture was warmed toroom temperature, and stirring was continued for 6 h. Solvent wasevaporated under vacuum, and the residue obtained was purified by flashchromatography using 7/3 petroleumether/ethylacetate as eluent tofurnish 141 mg of the product (12) (70% yield) as a gum.

[0065] H¹NMR (CDCl₃): ∂1.0 (m,1H, 6′CHH), 1.24 (s, 3H,CH₃), 1.27-1.38(m, 1H, 6′CHH), 1.55 (s, 3H, CH₃), 1.62 (dd, 1H, J=4.88, 9.77 Hz, 1′aH),3.34 (d, 1H, J=9.77 Hz, 5′CHH), 3.97 (d, 1H, J=9.77 Hz, 5′CHH), 4.50 (d,1H, J=6.84 Hz, 3′CH), 4.57-4.68 (qAB, 2H, J=12.7 Hz, ArCH₂), 5.17 (s,1H, 1′CH), 5.32 (d, 1H, J=6.84 Hz, 2′H), 7.27.4 (m, 5H, Ar), 8.63 (s,1H, 8CH).

[0066] (2R, 3R, 4R, 1′aR,1S)-2,3-(O-isopropylidine)-4-(methylenebenzyloxy)-1-(2-chloro-6-cyclopentylaminopurine-9-yl)bicyclo(3.1.0)hexane)(15):

[0067] To a solution of 12 (42 mg, 0.105 mmol) in methanol (2 ml) wasadded cyclopentylamine at room temperature, and stirring was continuedfor 6 hr for complete reaction. Solvent was removed under vacuum, andthe residue obtained was purified by flash column chromatography using7/3 petroleum ether/ethylacetate as eluent to furnish 45 mg of theproduct 15 (90% yield) as a gum.

[0068] H¹NMR(CDCl₃): δ0.92-0.96 (m, 1H. 6′CHH), 1.14-1.01 (m, IH,6′CHH), 1.23 (s, 3H, CH₃), 1.42-1.81 (m, 9H, 1′aH, 4CH₂), 1.54 (s, 3H,CH₃), 2.08-2.21 (m, 1H, N⁶ CH), 3.44 (d, 1H, J=9.76 Hz, 5′CHH), 3.90 (d,1H, J=9.76 Hz, 5′CHH), 4.51 (d, 1H, J=6.84 Hz, 3′CH), 4.57-4.67 (qAB,2H, J=12.7 Hz, ArCH₂), 5.04 (s, 1H, 1′CH), 5.32 (d, 1H, J=6.84 Hz,2′CH), 7.2-7.4 (m, 5H, Ar), 8.18 (s, 1H, 8CH).

[0069] (1′R, 2′R, 3′R, 4′R,1′aR)-2,3-(O-isopropylidine)-4-(methylenebenzyloxy)-1-(2-chloro-6-(3-idobenzylamino)purine-9-yl)bicyclo(3.1.0)hexane)(16) was synthesized in 70% yield by the same method as 15, except using3-iodobenzylamine hydrochloride and two equivalents of triethylamine.

[0070] H¹NMR(CDCl₃): δ0.87-0.91 (m, 1H, 6′CHH), 1.10-1.29 (m, 1H,6′CHH), 1.17 (s, 3H, CH₃), 1.42-1.56 (m, 1H, 1′aH), 1.47 (s, 3H, CH₃),3.37 (d, 1H, J=9.77 Hz, 5′CHH), 3.84 (d, 1H, J=9.77 Hz, 5′CHH), 4.44 (d,1H, J=6.84 Hz, 3′CH), 4.50-4.60 (qAB, 2H, J=11.72 Hz, ArCH₂), 4.70 (bs,1H, NH), 4.98 (s, 1H, 1′CH), 5.24 (d, 1H, J=6.84 Hz, 2′CH), 7.0 (t, 1H,J=7.82 Hz, ArH), 7.2-7.34 (m, 6H, ArH), 7.55 (d, 1H, J=7.82, ArH), 7.65(s, 1H, ArH), 8.08 (s, 1H, 8CH).

[0071] Pharmacological Analyses.

[0072] Materials

[0073] F-12 (Ham's) medium, fetal bovine serum (FBS) andpenicillin/streptomycin were from Gibco BRL (Gaithersburg, Md.).[¹²⁵I]AB-MECA (1000 Ci/mmol) and [³⁵S]guanosine 5′-(γ-thio)triphosphate(1000-1500 Ci/mmol) were from DuPont NEN (Boston, Mass.). Adenosinedeaminase (ADA) was from Boehringer Mannheim (Indianapolis, Ind.). Allother materials were from standard local sources and of the highestgrade commercially available.

[0074] Cell Culture and Membrane Preparation

[0075] CHO cells stably transfected with either human A₁ or A₃ receptors(gift of Dr. Gary Stiles and Dr. Mark Olah, Duke University MedicalCenter) were cultured as monolayers in medium supplemented with 10% afetal bovine serum. Cells were washed twice with 10 ml of ice-coldphosphate buffered saline, lysed in lysis buffer (10 mM Tris.HCI buffer,pH 7.4, containing 2 mm MgCI₂ and 0.5 mM EDTA), and homogenized in aPolytron homogenizer in the presence of 0.2 U/ml adenosine deaminase.The crude membranes were prepared by centrifuging the homogenate at1000×g for I0 min followed by centrifugation of the supernatant at40,000×g for 15 min. The pellet was washed once with the lysis bufferand recentrifuged at 40,000×g for 15 min. The final pellets wereresuspended in 50 mM Tris.HCl buffer, pH 7.4, containing I0 mM MgCI₂ and0. 1 mM EDTA and stored at −70° C.

[0076] Radioreceptor Binding

[0077] Determination of binding to adenosine A₁, A_(2A)and A_(2B)receptors was carried out as reported. Determination of A₃ adenosinereceptor binding was carried out using [¹²⁵I]AB-MECA. Briefly, aliquotsof crude transfected CHO cell membranes (approximately 40 μgprotein/tube) were incubated with 0.5 nM [¹²⁵I]AB-MECA, 10 mM MgC1₂, 2units/ml adenosine deaminase, 50 mM Tris.HCl (pH 7.4) at 37° C. for 60min. The total volume of the reaction mixture was 125 μl. Bound and freeligands were separated by rapid filtration of the reaction mixturethrough Whatman GF/B glass filters. The filters were immediately washedwith two 5 ml-portions of ice-cold 50 mM Tris.HCI buffer (pH 7.4). Theradioactivity bound to the filters was determined in a Beckman gammacounter. Specific binding was defined as the amount of the radioligandbound in the absence of competing ligand minus the amount of that boundin the presence of 100 μM NECA. Ki-values were calculated using theK_(d)for [¹²⁵I]AB-MECA binding of 0.56 nM.

[0078] Determination of [3′S]GTPγS Binding [³⁵S]GTPγS binding wasdetermined by the method of Lorenzen et al. The incubation mixturecontained in a total volume of 125 μl, 50 mM Tris.HCl (pH 7.4), 1 mMEDTA, 10 mM MgC1_(2,) 10 μM guanosine 5′-diphosphate, 1 mMdithiothreitol, 100 mM NaCl, 0.2 units/ml adenosine deaminase, 0.16 nM[³⁵S] GTPγS (about 50,000 cpm) and 0.5% BSA. The CHO cell membranesexpressing A₁ or A₃ receptors were preincubated with the above-mentionedassay mixture at 37° C. for 1 h and further incubated for 1 hr after theaddition of [³⁵S]GTPγS. Incubations were terminated by rapid filtrationof the samples through glass fiber filters (Whatman GF/B), followed bytwo 5 ml washes of the same buffer. After transferring the filters intoa vial containing 3 ml of scintillation cocktail, the radioactivity wasdetermined in a scintillation counter.

[0079] Data analysis. Analyses of saturation binding assays andconcentration-response curves were carried out using the GraphPad Prism(GraphPad Software Inc., San Diego, Calif.). Comparisons between groupswere carried out using the unpaired Student's test.

[0080] Results

[0081] Chemical Synthesis

[0082] The methanocarbocyclic 2′-deoxyadenosine analogues, shown belowin Table 1, in which a fused cyclopropane ring constrains thecyclopentane ring into a rigid envelope configuration of either a N- orS-conformation, were synthesized in a manner similar as shown above. TheN-methanocarba analogues of various N⁶-substituted adenosinederivatives, including cyclopentyl and iodobenzyl, in which the parentcompounds are potent and selective agonists at either A₁ or A₃receptors, respectively, were prepared. 2,6-Dichloropurine, 10, wascondensed with the cyclopentyl derivative, 11, using the Mitsunobureaction, followed by substitution at the 6-position and deprotection togive 8c or 9e. The 2-chloro substitution of compound 8c was removed bycatalytic reduction to give 6c. This allowed the incorporation in theN-configuration series of the 2-chloro modification of adenine, whichwas of interest for its effect on adenosine receptor affinity. AnN⁶-(3-iodobenzyl) group could also be introduced in eitheraristeromycin, 5b, or N-methanocarba-adenosine, 5c, by the Dimrothrearrangement, to give 7b and 7c.

[0083] Biological Activity

[0084] A pair of methanocarba analogues of adenosine, 5c and 5d,corresponding to N- and S-conformations of ribose, were tested inbinding assays, the results of which are shown in Table 1 below, at foursubtypes of adenosine receptors. The more synthetically challengingS-isomer (5d) was available only as the racemate and therefore wastested as such. At rat Al, rat A2A, and human A3 subtypes, theN-analogue proved to be of much higher affinity than the S-analogue. Atthe human A2B receptor, binding was carried out using [3H]ZM 241,385,however the affinity was too weak to establish selectivity for aspecific isomer. Affinity of N-methanocarba-adenosine, 5c, vs.adenosine, 5a, was particularly enhanced at the A3 receptor subtype, forwhich the ratio of affinities of N- to S-analogues was 150-fold.Although a poor substrate for adenosine deaminase (ADA), the bindingcurve for 5c was shifted in the presence of ADA, therefore the affinityvalues for 5c and 5d obtained in the absence of ADA are entered in Table1, below. The South confomer, 5d, is even a worse substrate of ADA(100-fold less) which explains why the curves in the presence andabsence of ADA for 5d are virtually the same. Aristeromycin, 5b, boundweakly to adenosine receptors, with slight selectivity for the A_(2A)subtype. Compound 5c was more potent than aristeromycin, 5b in bindingto A1 (4-fold) and A3 (4500-fold) adenosine receptors.

[0085] Compounds 6c and 8c are patterned after Al receptor-selectiveagonists, while compounds 7c and 9c are patterned after A3receptor-selective agonists. Compounds 6 and 7 are unsubstituted at the2-position, while compounds 8 and 9contain the potency enhancing2-chloro substituent. The N6-cyclopentyl N-methanocarba derivative, 6c,based on CPA, 6a, maintained high selectivity for Al receptors, althoughthe affinity of 6c at rat Al receptors was 3-fold less than for 6a. Inone series it was possible to compare ribose, cyclopentyl, andN-methanocarba derivatives having the same N6-substitution. TheN6-(3-iodobenzyl) derivative, 7c, based on a 5′-hydroxy analogue, 7a, ofIB-MECA, with a Ki value of 4.1 nM was 2.3-fold more potent at A3receptors than the ribose-containing parent. Thus, the selectivity of 7cfor human A3 versus rat AI receptors was 17-fold. The aristeromycinanalogue, 7b, was relatively weak in binding to adenosine receptors.

[0086] Among 2-chloro-substituted derivatives, the N-methanocarbaanalogue, 8c, was less potent at Al and A2A receptors than its parent2-chloro-N6-cyclopentyladenosine, 8a, and roughly equipotent at A3receptors. Thus, 8c was 53-fold selective in binding to rat Al vs. humanA3 receptors. The N-methanocarba analogue, 9c, of2-chloro-N6-(3iodobenzyl)adenosine, 9a, had Ki values (nM) of 141, 732,and 2.2 at Al, A2A, and A3 receptors, respectively. Thus, the 2-chlorogroup slightly enhanced affinity at A3 receptors, while reducingaffinity at Al receptors.

[0087] The receptor binding affinity upon replacement of ribose with theN-methanocarba moiety was best preserved for the A3 subtype, at whichdifferences were small. At Al receptors the loss of affinity forstructures 6-9 was between 3- and 8-fold. At A2A receptors the loss ofaffinity was between 6- and 34-fold;

[0088] The agonist-induced stimulation of binding of guanine nucleotidesto activated G-proteins has been used as a functional assay for avariety of receptors, including adenosine receptors. Binding of[³⁵S]GTP-γ-S was studied in membranes prepared from CHO cells stablyexpressing human A1 or A3 receptors (Table 2). The non-selectiveadenosine agonist NECA (5′-N-ethyluronamidoadenosine) caused aconcentration-dependent increase in the level of the guanine nucleotidebound. Compound 6c was highly selective and a full agonist at human Albut not rat Al receptors. Both 7c and 9c stimulated the binding of[³⁵S]GTP-γ-S, however the maximal stimulation was significantly lessthan that produced by either NECA or N6(3-iodobenzyl)adenosine, 7a, bothbeing full A3 agonists. Compounds 7c and 9c resulted in relativestimulation of [³⁵S]GTP-γ-S binding of only 45% and 22%, respectively,indicating that the efficacy of the N-methanocarba analogue at A3receptors was further reduced upon 2-chloro modification. The potency ofcompounds 7c and 9c, indicated by the EC50 values in this functionalassay, was greater than the potencies of either NECA or compound 7a(Table 2). Thus, the N-methanocarba N6-(3-iodobenzyl) analogues appearto be highly potent and selective partial agonists at human A3receptors. TABLE I Affinities of Adenosine Derivatives¹

K₁ (nM) or % displacement Compound R′ R RA₁ ^(a) RA_(2A) ^(b) hA₃ ^(c)1a H cyclopentyl  0.59   462  274 ± 20 CPA  240 (r) 1B H cyclopentyl 5.06 ± 0.51   6800 ± 1800  170 ± 51 1781 1c H cyclopentyl 5110 ± 790   15% at 10 μM 1783 2a H 3-iodobenzyl  20.0 ± 8.5  17.5 ± 0.5  9.5 ±1.4 (r) IB0-ADO,541 2b H 3-iodobenzyl  69.2 ± 9.8   601 ± 236 4.13 ±1.76 1743 3a Cl yclopentyl   0.6   950  237 (r) CCPA 3b Cl cyclopentyl 8.76 ± 0.81   3390 ± 520  466 ± 58 1761 3c Cl cyclopentyl 3600 ± 780   45 ± 5% at 100 μM 1782 4a Cl 3-iodobenzyl  18.5 ± 4.7  38.5 ± 2.01.41 ± 0.17 (r) 542 4b Cl 3-iodobenzyl  141 ± 22   732 ± 207 2.24 ± 1.451760 4c Cl 3-iodobenzyl 8730 ± 370 25,400 ± 3800 1784 Compound R₂ rA₁^(a) rA_(2A) ^(b) hA_(2b) ^(b) hA₃ ^(b) A₁/A₃ 5a H EStd. 10^(d) estd.30^(d) <10% at 100 μM estd. 1000(r)^(d,e) 100 5b H   6260 ± 730   2150 ±950 47,300 ± 10,600 20,000 ± 7900(r)^(e) 0.31 5c H   1680 ± 80 22,500 ±100 (h)^(e,f)    35 ± 2% at 50 μM^(f)   404 ± 70^(f) 4.2 5d H 15% at 100μM >100,000 (h)^(e,f)    20 ± 4% at 50 μM^(f) 62,500 ± 2900^(f) >1(racemic) 6a CP  1.50 ± 0.51   857 ± 163 21,200 ± 4300   274 ± 20,0.0055 240 (r)^(e) 6c CP  5.06 ± 0.51   6800 ± 1800   139k ± 19k   170 ±51 0.030 7a IB  20.0 ± 8.5  17.5 ± 0.5   3570 ± 100   9.5 ± 1.4(r)^(e)2.1 7b IB 25,900 ± 1600 <10% 100 μM n.d.   1960 ± 370 13 7c IB  69.2 ±9.8   601 ± 236 12,100 ± 1300  4.13 ± 1.76 17 8a CP  1.33 ± 0.19   605 ±154 20,400 ± 1200   237 (r)e 0.0056 8c CP  8.76 ± 0.81   3390 ± 520   27 ± 7% at 100 μM   466 ± 58 0.019 9a IB  18.5 ± 4.7  38.5 ± 2.0  5010 ± 1400  1.41 ± 0.17 (r)^(e) 13 9c IB   141 ± 22   732 ± 20741,000 ± 700  2.24 ± 1.45 63

[0089] TABLE II Effect of ligands to stimulate [³⁵S] GTPγS binding tomembranes of cells expressing the cloned hA₁AR or hA₃AR or in ratcerebral cortical membranes containing the A₁AR cloned hA₁AR % MaximalrA₁AR % Maximal cloned hA₁AR % Maximal Ligand EC₅₀ (nM)^(a)Stimulalion^(c) EC₅₀ (nM)^(a) Stimulation^(c) E₅₀ (nM)^(a)Stimulation^(c) NECA n.d. n.d. 155 ± 15  100 6a 4.15 ± 0.90 100  20.3 ±13.1 100 7980 ± 60  100 6c 21.5 ± 2.3  102 ± 1  100 ± 17 75 ± 6 >10,00014 ± 2% at 10 μM 7a 43.1 ± 10.4 91 ± 1 340 ± 98 95 ± 4 5.16 ± 0.71 1007b >10,000 5 ± 2% at 10 μM n.d. >10.000 15 ± 5% at 10 μM 7c 218 ± 18  86± 2  940 ± 114 55 ± 5 0.70 ± 0.16 45.3 ± 6.8 8c 31.2 ± 3.3  97 ± 1 145 ±35 96 ± 2 n.d. 9c 142 ± 24  91 ± 1 684 ± 75 48 ± 3 0.67 ± 0.19 22.0 ±2.8

[0090] Discussion

[0091] Nearly all of the thousands of known adenosine agonists arederivatives of adenosine. Although molecular modeling of adenosineagonists has been carried out, there has been no direct evidence fromthis for a conformational preference of the ribose ring in the receptorbinding site. In the present study, methanocarba-adenosine analogueshave defined the role of sugar puckering in stabilizing the activereceptor-bound conformation. The S-methanocarba analogue of adenosine,5d, was only weakly active, presumably because of a disfavoredconformation that decreases receptor binding. In contrast, themethanocarba analogues constrained in the N-conformation, e.g. 5c-9c,displayed high receptor affinity, particularly at the A3 receptor. Inbinding assays at Al , A2A, and A3 receptors, N-methanocarba-adenosineproved to be of higher affinity than the S-analogue, with anN:S-affinity ratio of 150 at the human A3 receptor. Thus, the biologicalpotency and efficacy of this series of nucleosides appears to be highlydependent on ring puckering, which in turn would influence theorientation of the hydroxyl groups within the receptor binding site.

[0092] The structure activity relationship (SAR) of adenosine agonistsindicates that the ribose ring oxygen may be substituted with carbon, asin 5b and 7b, however much affinity is lost. As demonstrated with thearisteromycin derivative, 7b, simple carbocyclic substitution of theribose moiety of otherwise potent, N6-subsituted adenosine agonistsgreatly diminishes affinity, even in comparison to aristeromycin, 5b.

[0093] In comparison to the ribose analogues, the N-methanocarbaN6-subsituted adenosine agonists were of comparable affinity at A3receptors, but less potent at Al, A2A, and A2B receptors. TheN-methanocarba N6-cyclopentyl derivatives were Al receptor-selective andmaintained high efficacy at human recombinant but not rat brain A1receptors; as indicated by stimulation of binding of [³⁵S]GTPγS. Thismay be related to either species differences or heterogeneity of Gproteins, since the degree of agonist efficacy of a given compound maybe highly dependent on the receptor-associated G protein. N-MethanocarbaN6-(3-iodobenzyl)adenosine and the 2-chloro derivative had Ki values of4.1 and 2.2 nM at A3 receptors, respectively, and were selective partialagonists. As for the ribose parents, additional 2-chloro substitutionwas favorable for receptor selectivity. However, unlike the riboseforms, efficacy was reduced in N6-(3-iodobenzyl) analogues, such thatpartial A3 receptor agonists 7c and 9c were produced.

[0094] Partial agonists are possibly more desirable than full agonistsas therapeutic agents due to potentially reduced side effects in theformer. Partial agonists may display in vivo specificity for sites atwhich spare receptors are present, and the drug would therefore behavewith apparent “full” efficacy. Thus, for compounds 7c and 9c, partialagonism combined with unprecedented functional potency at A3 receptors(<1 nM) may give rise to tissue selectivity.

[0095] Thus, at least three of the four adenosine receptors favor theN-conformation. For another member of the GPCR superfamily, the P2Y1receptor, we recently reported that the ribose N-conformation of adeninenucleotides also appears to be preferred at the receptor binding site.Thus, the P1 and at least one of the P2 purinoceptors share thepreference for the N-conformation. This may suggest a common motif ofbinding of nucleoside moieties among these GPCRS. The insights of thisconformational preference may be utilized in simulated docking ofadenosine agonists in a putative receptor binding site and to designeven more potent and selective agents.

[0096] At the binding site of ADA, the N-isomer is also preferred,although the carbocyclic adenosine analogues are relatively poorsubstrates (relative rates of deamination are: 5a, 100; 5b, 0.99; 5c,0.58; 5d, 0.010, N6-substituted analogues, such as 6c-9c, would not beexpected to be substrates for ADA. Other enzymes, such as HIV reversetranscriptase and Herpes thymidine kinase (HSV-1 TK) are also able todiscriminate between the two antipodal conformations of restrictedmethanocarba thymidine analogues.

[0097] In conclusion, we have found that the introduction of amethano-carbocyclic modification of the ribose ring of purine agonistsrepresents a general approach for the enhancement of pharmacodynamic andbecause of the absence of the glycosyl bond, potentially ofpharmacokinetic properties. This approach could therefore be applied tothe development of cardioprotective, cerebroprotective, andanti-inflammatory agents.

EXAMPLE 2

[0098] Introduction

[0099] P2 receptors, which are activated by purine and/or pyrimidinenucleotides, consist of two families: G protein-coupled receptors termedP2Y, of which 5 mammalian subtypes have been cloned, and ligand-gatedcation channels termed P2X, of which 7 mammalian subtypes have beencloned. The P2Y₁, receptor, which is present in the heart, skeletal andvarious smooth muscles, prostate, ovary, and brain, was the first P2subtype to be cloned. The nomenclature of P2 receptors and their variousligand specificities is well established.

[0100] Nucleotide agonists binding at P2Y₁, receptors induce activationof phospholipase C (PLC), which generates inositol phosphates anddiacylglycerol from phosphatidyl inositol-(4,5)-bisphosphate, leading toa rise in intracellular calcium. A P2Y₁ receptor antagonist may havepotential as an anti-thrombotic agent, while a selective P2Y₁ receptoragonist may have potential as an anti-hypertensive or anti-diabeticagent.

[0101] Recently, progress in the synthesis of selective P2 receptorantagonists has occurred. Adenosine 3′,5′- and 2′,5′-bisphosphates wererecently shown to be selective antagonists or partial agonists at P2Y₁,receptors, and other classes of P2 antagonists include pyridoxalphosphate derivatives, isoquinolines, large aromatic sulfonates relatedto the trypanocidal drug suramin and various dyestuffs, and2′,3,-nitrophenylnucleotide derivatives. Synthesis of analogues ofadenosine bisphosphates has resulted inN6-methyl-2′-deoxyadenosine-3′,5′-bisphosphate (1a, MRS 2179), acompetitive antagonist at human and turkey P2Y₁ receptors, with a KBvalue of approximately 100 Nm. The presence of an N⁶-methyl group andthe absence of a 2′-hydroxyl group both enhanced affinity and decreasedagonist efficacy, thus resulting in a pure antagonist at both turkey andhuman P2Y₁, receptor. The corresponding 2-C1 analogue (1b, MRS 2216) wasslightly more potent than 1a as an antagonist at turkey P2Y₁ receptors,with an IC₅₀ value of 0.22 μM in blocking the effects of 10 nm2-methylthioadenosine-5′diphosphate (2-MeSADP). MRS2179 (compound1a )was inactive at P2Y₂, P2Y₄, and P2Y₆, subtypes, at the adenylylcyclase-linked P2Y receptor in C6 glioma cells and at a novel avian P2Yreceptor that inhibits adenylyl cyclase. However, the selectivity ofthis series of nucleotides for the P2Y₁ receptor is not absolute, since1a also displayed considerable activity at P2X₁, receptors (EC₅₀ 1.2μM), but not at P2Y₂₋₄ receptors.

[0102] In order to move away from the nucleotide structure of 1a andthereby increase biological stability and selectivity for the receptorsin the present study, further structural modifications of the ribosemoiety have been carried out. We have explored the SAR of these twoseries and introduced major modifications of the ribose moiety. Thesemodifications include fixing the ring pucker conformation in thecarbocyclic series using a bridging cyclopropane ring, ring enlargementwith introduction of a nitrogen atom, and ring contraction. Results

[0103] Chemical Synthesis

[0104] The methanocarbocyclic 2′-deoxyadenosine analogues in which thefused cyclopropane ring fixes the conformation of the carbocyclicnucleoside into a rigid northern or southern envelope conformation, asdefined in the pseudoroational cycle, were synthesized as precursors ofnucleotides 4 and 5 by the general approach of Marquez and coworkers.Again, the N⁶-methyl group was introduced by the Dimroth rearrangement,as shown below.

[0105] Position adenine modifications were further introduced in theN-configuration series as shown below.

[0106] Biological Activity

[0107] Adenine nucleotides markedly stimulate inositol lipid hydrolysisby phospholipase C in turkey erythrocyte membranes, through activationof a P2Y₁, receptor, The agonist used in screening these analogues,2-MeSADP, has a higher potency than the corresponding triphosphate forstimulation of inositol phosphate accumulation in membranes isolatedfrom [³H]inositol-labeled turkey erythrocytes.

[0108] The deoxyadenosine bisphosphate nucleotide analogues prepared inthe present study were tested separately for agonist and antagonistactivity in the PLC assay at the P2Y₁ receptor in turkey erythrocytemembranes, and the results are reported in Table 3.Concentration-response curves were determined for each compound aloneand in combination with 10 nM 2-MeSADP.

[0109] Marquez and coworkers have introduced the concept ofring-constrained carbocyclic nucleoside analogues, based on cyclopentanerings constrained in the N-(Northern) and S-(Southern) conformations byfusion with a cyclopropane (methanocarba) ring. In the presnet studiesthe series of ring-constrained N-methanocarba derivatives, the 6-NH₂analogues, 4a was a pure agonist of EC₅₀152 nM and 88-fold more potentthan the corresponding S-isomer, 5, also an agonist. Thus, the ribosering N-conformation appeared to be favored in recognition at P2Y₁receptors. The N⁶-methy- and 2-chloro-N⁶-methyl-N-methanocarbaanalogues, 4b and 4c, were antagonists having IC₅₀ values of 276 and 53nM, respectively.

[0110] Molecular Modeling.

[0111] To better understand the role of the sugar puckering on the humanP2Y₁ agonist and antagonists activities, we carried out a molecularmodeling study of this new generation of ribose-modified ligands. Suchmodifications include cyclopentyl rings constrained in the N- andS-conformations with cyclopropyl (methanocarba) groups, six-memberedrings (morpholino and anhydrohexitol analogues), and cyclobutylnucleotides. We have recently developed a model of the human P2Y₁receptor, using rhodopsin as a template, by adapting a facile method tosimulate the reorganization of the native receptor structure induced bythe ligand coordination (cross-docking procedure). Details of the modelbuilding are given in the Experimental Section. We have also reportedthe hypothetical molecular basis for recognition by human P2Y₁,receptors of the natural ligand ATP and the new potent, competitiveantagonist 2′-deoxy-N⁶-methyladenosine-3′,5′-bisphosphate. Both ATP and1a are present in the hypothetical binding site with a N-sugar ringconformation. In the present work, the sterically constrained N- andS-methanocarba agonist analogues, 4a and 5, respectively, were dockedinto the putative binding site of our previously reported P2Y₁ receptormodel. According to their structural similarity, the cross-dockingprocedure demonstrated that the receptor architecture found for bindingthe ATP and 1a was energetically appropriate also for the binding ofboth 4a and 5. However, N-methanocarba/P2Y₁ complex appeared more stableby approximately 20 kcal/mol than S-methanocarba/P2Y₁, complex. In thelowest energy docked complex of N-methanocarba agonist in the proposedligand binding cavity the side chain of Gln307 is within hydrogenbonding distance of the N⁶ atom at 1.8 Å, and the side chain of Ser314is positioned at 2.0Å from the N¹ atom and at 3.4 Å from the N⁶ of thepurine ring. As already reported, another three amino acids areimportant for the coordination of the phosphate groups in theantagonist: Arg128, Lys280 and Arg310. Lys280 may interact directly withboth 3′-5′-phosphates (1.7 Å, O3′ and 1.7 Å, O5′), whereas Arg128 andArg310 are within ionic coupling range to both the O2 and O3 atoms ofthe 5′-phosphate. In molecular modeling studies poor superimposition(rms=1.447),between the N- and S-methanocarba agonist analogues has beenfound inside the receptor binding domain. In particular,. the adeninemoiety and 5′ phosphate of the S-methanocarba derivative are shifted outposition relative to with the N-methanocarba isomer, decreasing thestability of the S-methanocarba/PSY₁ complex. This fact might becorrelated with the difference of their biological activity as seen inTable 4 below.

[0112] Using the information that a common binding site could behypothesized among these deoxyadenosine bisphosphate analogues, asuperimposition analysis of the energy-minimized of the more potentantagonists has been performed. In this analysis we have used 1a as areference compound, and we have defined three matching pairs of atoms,corresponding to N¹ atom of the purine ring and the P atoms of both 3′and 5′ phosphate groups, to carry out the superimposition analysis. Asreported in Table 4, acceptable RMS values have been obtained for allthe antagonists compared with the 1a structure. As shown in FIG. 4A,this superimposition study suggested that the two phosphate groups mayoccupy a common receptor regions, and a general pharmacophore model forbisphosphate antagonists binding to the human PSY₁ receptor can beextrapolated.

[0113] Discussion

[0114] In conclusion the present study has identified newpharmacological probes of PSY₁, receptors, including full agonists,partial agonists, and antagonists. The SAR of 1a indicates that theribose ring oxygen may be readily substituted with carbon. Furthermore,analogues of constrained conformation, e.g. the methanocarba analogues,display enhanced receptor affinity. Additional 2-chloro and N⁶-methylsubstitution is favorable for affinity at PSY₁ receptors, and nearlypure antagonism is maintained provided that the N⁶-methyl group ispresent.

[0115] Thus, the biological potency and efficacy of this series ofbisphosphates appears to be highly dependent on subtle conformationalfactors, which would influence the orientation of the phosphate groupswithin the receptor binding site.

[0116] The sugar moiety of nucleosides and nucleotides in solution isknown to exist in a rapid, dynamic equilibrium between extreme2-exo/3′-endo (N-) and 2′-endo/3′-exo (S-) conformations as defined inthe pseudorotational cycle. While the energy gap between N- andS-conformation is in the neighborhood of 4kcal/mol, such a disparity canexplain the difference between micromolar and nanomolar bindingaffinities. Using a molecular modeling approach, we have analyzed thesugar conformational requirements for a new class of bisphosphateligands binding to the human PSY₁ receptor. As experimentally shown, theribose ring Northern conformation appeared to be favored in recognitionat human PSY₁ receptor (see Table 4). We have found new support to ourrecently presented hypothesis in which three important recognitionregions are present in the bisphosphate molecular structures; The N¹atom of the purine ring and the P atoms of both 3′ and 5′ phosphategroups. The N-conformation seems to be essential to maximize theelectrostatic interactions between the negatively charged phosphates andthe positively charged amino acids present in the receptor bindingcleft, as well Arg128, Lys280, and Arg310.

[0117] Interestingly, the electrostatic contacts also appear to becrucial for the recognition of bisphosphate antagonists. Usingsuperimposition analysis, a general pharmacophore model for thebisphosphate antagonists binding to the PSY₁ receptor has been proposed.According to the pharmacophore map, recognition of the bisphosphatesantagonists at a common region inside the receptor binding site and,consequently, a common electrostatic potential profile is possible. Aswell for the agonists, the Northern conformation seems to be essentialto maximize the electrostatic interactions between the negativelycharged phosphates and the positively charged amino acids presents inthe receptor binding cleft. As we predicted using the previouslyreported PSY₁ receptor model, sugar moiety does not seen to be crucialfor the ligand recognition process.

[0118] As already described, the simple addition of the N⁶-methyl groupin several cases converted pure agonists to antagonists. From apharmacological point of view, this is really a unique situation. Withthe addition of the N⁶-methyl group it is not possible to have a doublehydrogen-bonding interaction and, consequently, the activation pathwayis blocked. However, for all the N⁶-methyl antagonists the possibilityto participate in at least one of the two possible hydrogen bondsappears to be very important for the increase in affinity at the PSY₁receptor.

[0119] Chemical Synthesis

[0120] Nucleosides and synthetic reagents were purchased from SigmaChemical Co. (St. Louis, Mo.) and Aldrich (St. Louis, Mo.).6-Chloro-2′-deoxypurine riboside was obtained from Sigma. Several2′-deoxynucleosides, including an anhydrohexitol-adenine nucleoside and2′-deoxyaristeromycin were also synthesized.

[0121] Purity of compounds was checked using a Hewlett-Packard 1090 HPLCapparatus equipped with an SMT OD-5-60 RP-C18 analytical column (250×4.6mm; Separation Methods Technologies, Inc., Newark, Del.) in two solventsystems. System A: Linear gradient solvent system: 0.1 M TEAA/CH₃CN from95/5 to 40/60 in 20 min and the flow rate was of 1 mL/min. System B:linear gradient solvent system: 5 mM TBAP/CH₃CN from 80/20 to 40/60 in20 min and the flow rate was of 1 mL/min. Peaks were detected by UVabsorption using a diode array detector. All derivatives showed morethan 95% purity in the HPLC systems.

[0122] Purification of most of the nucleotide analogues, for biologicaltesting was carried out on DEAE-A25 Sephadex columns as described above.However, compounds 7b and 8a-c required HPLC purification (system a,semi-preparative C18 column) of the reaction mixtures.

[0123] General Procedure of Phosphorylation.

[0124] Method A: The nucleoside (0.1 mmol) and Proton Sponges® (107 mg,0.5 mmol) were dried for several h in high vacuum at room temperatureand then suspended in 2 mL of trimethyl phosphate. Phosphorousoxychloride (Aldrich, 37 μL, 0.4 mmol) was added, and the mixture wasstirred for 1 h at 0° C. The reaction was monitored by analytical HPLC(eluting with a gradient consisting of buffer: CH₃CN in the ratio 95:5to 40:60, in which the buffer was 0.1 M triethylammonium acetate (TEAA);elution time was 20 min; flow rate was 1 mL/min; column was SMT OD -5-60RP-C18; detector was by UV in the E_(max) range of 260-300 nm). Thereaction was quenched by adding 2 mL of triethylammonium bicarbonatebuffer and 3 mL of water. The mixture was subsequently frozen andlyophilized. Purification was performed on an ion-exchange column packedwith Sephadex-DEAE A-25 resin, a linear gradient (0.01 to 0.5 M) of 0.5M ammonium bicarbonate was applied as the mobile phase, and UV and HPLCwere used to monitor the elution. All nucleotide bisphosphates werecollected, frozen and lyophilized as the ammonium salts. All synthesizedcompounds gave correct molecular mass (high resolution FAB) and showedmore than 95% purity (HPLC, retention times are reported in Table 4).

[0125] Method B: Nucleoside (0.1 mmol) dried for several h in highvacuum at room temperature was dissolved in 2 mL of dry THF. Lithiumdiisopropylamide solution (Aldrich, 2.0 M in THF, 0.4 mmol) was addedslowly at −78° C. After 15 min tetrabenzyl pyrophosphate (Aldrich, 0.4mmol) was added and the mixture was stirred for 30-60 min at −78° C. Thereaction mixture was warmed to 0° C.-rt and stirred for an additionperiod ranging from 2h to 24h. Chromatographic purification (pTLC,CHCl₃:CH₃OH(10:1) gave the tetrabenzyl phosphorylated compound. Thiscompound (20 mg) was dissolved in a mixture of methanol (2 mL) and water(1 mL) and hydrogenated over a 10% Pd-on-C catalyst (10 mg) at rt for 62h. The catalyst was removed by filtration and the methanol wasevaporated. The residue was treated with ammonium bicarbonate solutionand subsequently frozen and lyophilized. Purification, if necessary, wasby the same procedure as in method A.

[0126] (N-Methanocarba-2′-deoxyadenosine-3′,5′-bis(diammoniumphosphate): (4a)[(IR,2S,4S,5S)-1-[(phosphato)methyl]-4-(6-aminopurin-9-yl) bicyclo[3.1.0]-hexane-2-phosphate tetraammonium salt]

[0127] Starting from 16 mg (0.06 mmol) of(N)-methanocarba-2′deoxyadenosine and following the generalphosphorylation procedure A we obtained 1.8 mg (0.0037 mmol, 5.5% yield)of the desired compound.

[0128]¹H-NMR (D₂O) ∂0.90 (1H, m, CH₂-6), 1.10 (1 H, m CH₂6′), 1.82 (1H,m, CH-5), 1.91 (1H, m, CH₂-3′) 2.23 (1H, m, CH₂-3′), 3.49 (1H, d, J=11.7Hz, CH₂-OH), 4.16 (1H, d, J=6.9 Hz, CH₂-2′), 8.39 (1H, s, H-2), 8.54(1H, s, H-8).

[0129]³¹P-NMR (D₂O) ∂0.43 (s, 5′P); −0.19 (S, 3′P).

[0130] (N)-Methanocarba-N⁶-methyl-2′deoxyadenosine-3′,5′-bis(diammoniumphosphate) (4b)

[0131] (IR,2S,4S,5S) -1-[(phosphato)methyl]-4-(6-methylaminopurin-9-yl)bicyclo [3.1.0]-hexane-2-phosphate tetraammoniun salt]

[0132] 13.5 mg (0.0170 mmol) of compound 18 was converted to thecorresponding phosphoric acid analog using hydrogenation following thegeneral procedure B. Purification was performed on an ion-exchangecolumn packed with Sephadex-DEAE A-25 resin, linear gradient (0.01 to0.5 M) of 0.5 M ammonium bicarbonate was applied as the elan to give 3.0mg (0.0060 mmol, 35.3% yield) of the desired compound.

[0133]¹H-NMR (D₂P) ∂0.93-0.98 (1H, m, CH₂-6′), 1.17 (1H, m, CH2-6′),1.86-1.88 (1, m, CH5′), 1.94-1.98 (1H, m, CH₂-3′), 2.23-2.31 (1H, m,CH₂-3′), 3.09 (3H, bs, N⁶-CH₃), 3.61-3.64 (1H, m, CH₂OH), 4.51-4.55 (1H, m, CH₂OH), 5.01-5.03 (1H, m,, CH-4′), 5.19-5.21 (1H, m, CH-2′), 8.22(1H s, H-2), 8.51 (1H, s, H-8). 31P-NMR (D₂O) ∂1.26, 1.92 (2s, 3′-P,5′-P).

[0134](N)-Methanocarba-N⁶-methyl-2-chloro-2′-deoxyadenosine-3′,5′-bis(diammoniumphosphate) (4c)

[0135][(1R,2S,4S,5S)-1[(phosphato)methyl]-4-(2-chloro-6-aminopurin-9-yl)bicyclo [3.1.0]-hexane-2-phosphate tetraammonium salt]

[0136] The nucleoside, compound 23, reacted with tetrabenzylpyrophosphate, as in Method B, followed by an alternative deprotectionprocedure. Starting from 10 mg (0.0323 mmol) of(N)-methanorcarba-N⁶-methyl-2-chloro-2′-deoxyadenosine and following thegeneral phosphorylation procedure (Method B) we obtained 9.5 mg (0.0114mmol, 35.3% yield) of the desired compound,(N)-methanocarba-N⁶-methyl-2-chloro-2′-deoxyadenosine-3′,5′-bis(dibenzyl phosphate).

[0137] 1H-NMR (CDCl₃) ∂0.75-0.81 (H, m, CH₂-6′), 103-1.08 (1H, m,CH₂-6′), 1.49-1.51 (1H, m, CH-5′), 1.84-1.94 (1H, m, CH₂-3′), 1.99-2.10(1H, m, C₂-3′), 3.12 (3H, bs, N⁶-CH₃), 4.11-4.20 (1H, m, CH₂OH),4.50-4.55 (H, m, CH₂OH), 4.90-4.98, (8H, m,—OCH₂) 4.99-5.01 (1H, m,CH-4′), 5.23-5.30 (1H, m, CH-2′), 5.90 (1H, BS, NH), 7.20-7.29 (20H, m,C₆H₅), 7.82 (1H, s, H-8) ³¹P-NMR (D₂O) ∂-0.58 (s,5′P); −1.06 (s,3′P).MS(CI-NH₃) (M+1) 830 HRMS (FAB-) (M+Cs) Calcd. 962.1252;Found 962, 1252

[0138]9.5 mg (0.0114 mmol) of the tetrabenzyl-protected intermediateadded to dry CH₂Cl₂ (1.0 mL) was cooled to −78° C. under argon andtreated with 100 μL of boron trichloride solution (1M in CH₂CI₂) and 100μL of anisole. The reaction mixture was stirred for 12 hr at 0° C. to rtand extracted with triethylamine solution. Purification was performed onan ion-exchange column packed with Sephadex-DEAE A-25 resin, lineargradient (0.01 to 0.5 M) of 0.5 M ammonium bicarbonate was applied asthe eluent to give 0.4 mg (0.0007 mmol, 6.52 yield) of the desiredcompound 4c.

[0139]¹H-NMR (D₂O) ∂0.91-0.96 (1H, m, CH₂-6′), 1.12-1.16 (1H, m,CH₂-6′), 1.80-1.84 (1H, m, CH-5′), 1.85-1.98 (1H, m, CH₂-3′), 2.20-2.50(1H, m, CH₂-3′), 3.08 (3H, bs, N⁶-CH₃), 3-57-3.60 (1H m, CH₂OH),4.52-4.67 (1H, m, CH₂OH), 4.94-4.96 (1H, m, CH-4′), 5.18-5.21 (1H, m,CH-2′), 8.52 (1H, s,H-8) ³¹P-NMR (D₂O) ∂1.82, 2.52 (2s, 3′-P, 5′P)

[0140] (S) -Methanocarba-2′, deoxyadeonosine-3′,5′-bis (diamnoniumphosphate (5) [(1S,3S,4R,5S)-4-[(phosphato)methyl]-1-(6-aminopurin-9-yl)bicyclo [3.1.0]-hexane-3-phosphate tetraammoxium salt]

[0141] Starting from 16 mg (0.06 mmol) of(S)-methanocarba-2′deoxyadenosine and following the generalphosphorylation procedure A, we obtained 2.1 mg (0.0043 mmol, 7.55yield) of the desired compound 5.

[0142]¹-NMR (D₂O) ∂1.36 (1H, m, CH₂-6′), 1.53 (1H, t, J=4.8 Hz, CH₂-6′),2.05 (1H, m, CH₂-5′), 2.30 (1H, m, CH-4′), 2.46 (2H, m, CH₂-2′), 3.97(2H, m, CH₂OH), 4.45 (1H, d, j=6.6 Hz, CH-3′), 8.16 (1H, s, H-2), 8.30(1H, s, H-8). ³¹ P-NMR (D₂O) ∂0.85 (bs, 5′P); 0.31 (bs, 3′p).

[0143] [(1S, 3S,4R,5S)-1-[(Hydroxy)methyl]-2-hydroxy-4-(6-methylaminopurin-9-lyl)bicyclo [3.1.0]-hexane (17b)

[0144] The Dimroth rearrangement (Scheme 2) was carried out on(N)-methanocarba-2′-deoxyadenosine. Specifically, the(N)-methanorcarba-2′-deoxyadenosine (17a, 50.0 mg, 0.191 mmol) washeated at 40° C. with methyl iodide (71.5 μL, 1.15 mmol) in dry DMF (2.0mL) for 48 h. The solvent was evaporated under reduced pressure, and theresidue was heated at 90° C. with ammonium hydroxide (4.0 mL) for 4 h.The water was evaporated, and the residue was purified by pTLC usingMeOH; CHCl₃ (1:9) to afford compound 17b as a colorless solid (40 mg,0.15 mmol, 76%).

[0145]¹H-NMR (CD₃OD) ∂0.77.-0.81 (1H, m, CH₂-6′), 1.03-1.07 (1H, m,CH₂-6′), 1.68-1.72 (1H, m, CH-5′), 1.79-1.89 (1H, m, CH₂-3′), 2.00-2.07(1H, m, CH₂ -3′), 3.12 (3H, bs, N⁶-CH₃), 3-33 (1H, d, J=CH₂OH), 4.29(1H, d, J=11.7 Hz, CH₂OH), 4.89-4.92 (1H, m, CH-4′); 5.02 (1H, d, J=6.9Hz, CH-2′), 8.24 (1H, s,H-2), 8.49 (1H, s, H-8). MS(Cl-NH₃): 276 (M+1)830 HRMS(FAB-) (M+Cs) Calcd. 275.1382; Found 275.1389.

[0146](N)-Methanocarba-N⁶-methyl-2′-deoxyadenosine-3′,5′-bis(dibenzylphosphate)(18)

[0147] [(1S,2S, 42,5S)-1-[(dibenzylphosphato)methyl]-4-(6-methylaminopurin-9-yl) bicyclo[3.1.0]-hexane-2-dibenzylphosphate]

[0148] Starting from 20.0 mg (0.0726′mmol) ofN-methanorcarba-N⁶-methyl-2′-deoxyadenosine 17b and following thegeneral phosphorylation procedure (Method B we obtained 13.5 mg (0.0170mmol, 23.4% yield) of the desired protected intermediate, 18 as shown inScheme 2.

[0149]¹H-NMR (CDCl₃) ∂0.73-0.78 (1H, m CH₂-6′), 0.94-0.98 (1H, m,CH₂-6′), 1.53-1.54 (1H, m, CH-5′), 1.81-1.91 (1H, m, CH₂-3′), 2.05-2.13(1H, m, CH₂-3′), 3.15 (3H, bs, N⁶-CH₃), 3-70-3.83 (1H, m, CH₂OP),4.49-4.55 (1H, m, CH₂OP), 4.89-5.00(8H, m, OCH-₂), 5.02-5.06 (1H, m,CH-4′), 5.27-5.32 (1H, m, CH-2′), 5.86 (1H, bs, NH), 7.21-7.23 (20H, m,C₆H₅), 7.86 (1H, s, H-2), 8.31 (1H, s, H-8). ³¹P-NMR (D₂O) ∂-0.56, −1.05(2s, 3′-P, 5′P) HRMS (FAB-) (M-Cs) Calcd. 928.1641; Found 928.1700.

[0150][(1S,2S,42,5S)-1-[(Benzyloxy)methyl]-2-benzyloxy-4-(2-6-dichloropurin-9-yl)bicyclo [3.1.0]-hexane (21)

[0151] To an ice cold solution of triphenylphosphine (278 mg, 1.06 mmol)in dry THF (2 mL) was added diethylazadicarboxylate (170 μL, 1.06 mmol)dropwise under a nitrogen atmosphere, and the mixture was stirred for 20min until the solution turned red orange (Scheme 3). This mixture wasadded dropwise to a cold stirred mixture of the starting alcohol (135mg, 0.417 mmol) and 2.6-dichloropurine (157 mg, 0.883 mmol) under anitrogen atmosphere. The reaction mixture was stirred in an ice bath for30 min and then allowed to warm to room temperature, and stirringcontinued for 12 h. Solvent was removed by nitrogen purge, and theresidue was purified by pTLC using EtOAc: petroleum ether (1:1) toafford a thick liquid (132 mg, 0.263 mmol, 64%).

[0152]¹H NMR: (CD₃OD) δ0.85 (m, 1H), 1.13 (m, 1H ), 1.59 (m, 1H), 1.68(m, 1H), 2.06 (m, 1H), 3.17 (d, J=10.8 Hz, 1H), 4.11-4.57 (m, 5H), 5.20(d, J=6.9 Hz, 1H ), 6.6 (bs, 1H), 7.23-7.37 (m, 10H), 8.98 (s, 1H). MS:(EI) 494 (M+).

[0153][(lR,2S,4S,5S)-1-[(Benzyloxy)methyl]-2-benzyloxy-4-(2-chloro-6-methylaminopurin-9-yl)bicyclo [3.1.0]-hexane (22)

[0154] Compound 21 (100 mg, 0.202 mmol) was dissolved in methylamine inmethanol (30% solution, 3mL) and was stirred at rt for 12 h under anitrogen atmosphere. The solvent was evaporated, and the crude productwas purified by pTLC using EtOAc: petroleum ether (6:4) to afford 22 asa light yellow solid (86 mg, 0.176 mmol, 88%).

[0155]¹H NMR: (CD₃OD) δ8 0.70 (m, 1H), 1.06 (m, 1H), 1.50 (m, 1H), 1.76(m, 1H), 1.96 (m, 1H ), 3.01 (s, 3H),, 3.08 (m, 2H), 4.03 (m, 4H), 4.45(bs, 1H), 5.02 (bs, 1H), 8.38 (s, 1H ). MS: (Cl): 490 (M+1).

[0156][(1R,2S,42,5s)-1-[(Hydroxy)methyl]-2-hydroxy-4-(2-chloro-6-methylaminopurin-9-yl) bicyclo [3.1.0]-hexane (23)

[0157] Compound 22 (40 mg 0.0816 mmol) was dissolved in dry CH₂Cl₂ (1.0mL), and hydrogenated using BCl₃ (1M in CH₂Cl₂, 175 μL) for 50 min at−78° C. under argon. The solvent was evaporated, and the crude productwas purified by pTLC using CHCl₃: MeOH (10:1) to afford 23 as a lightyellow solid (10.0 mg, 0.0323 mmol, 39.6%).

[0158]¹H NMR: (CD₃OD) ∂0.77-0.81 (1H, m, CH₂-6′), 1.02-1.05 (1H , m,CH₂-6′), 1.65-1.68 (1H, m, CH-5′), 1.78-1.91 (1H, m, CH₂-3′), 1.99-2.07(1H, m, CH₂-3′), 3.08 (3H, bs, N⁶-CH₃), 3.37 (1H, d, J=11.7 Hz, CH₂OH),4.27 (1H, d, J=11.7 Hz, CH₂OH), 4.89-4.91 (1H, m, CH-4), 4.97 (1H, d,J=6.8 Hz, CH-2′), 8.46 (1H, s, H-8).

[0159] MS: (CI-NH₃): 310 (M+1), HRMS (FAB-): Calcd 309.0992, Found309.0991.

[0160] Pharmacological Analyses.

[0161] P2Y₁ receptor promoted stimulation of inositol phosphateformation by adenine nucleotide analogues was measured in turkeyerythrocyte membranes as previously described. The K_(0.5) values wereaveraged from 3-8 independently determined concentration-effect curvesfor each compound.. Briefly, 1 mL of washed turkey erythrocytes wasincubated in inositol-free medium (DMEM; Gibco, Gaithersburg Md.) with0.5 mCi of 2-[³H]myo-inositol (20Ci/mmol: American RadiolabelledChemicals, Inc., St. Louis Mo.) for 18-24 h in a humidified atmosphereof 95% air/5% CO₂ at 37° C. Erythrocyte ghosts were prepared by rapidlysis in hypotonic buffer (5 mM sodium phosphate, pH 7.4, 5 mM MgCI₂,1mM EGTA) as described. Phospholipase C activity was measured in 25 μLof [³H] inositol-labeled ghosts (approximately 175 μg of protein,200-500,000 cpm/assay) in a medium containing 424 μM CaCl₂, 0.91 mMMgSO₄, 2 mM EGTA, 115 mM KCl, 5 mM KH₂PO₄, and 10 mM Hepes pH 7.0.Assays (200 μL final volume) contained 1 μM GTPγS and the indicatedconcentrations of nucleotide analogues. Ghosts were incubated at 30° C.for 5 min, and total [³H]inositol phosphates were quantitated by anionexchange chromatography as previously described.^(7,36)

[0162] Data Analysis.

[0163] Agonist potencies were calculated using a four-parameter logisticequation and the GraphPad software package (GraphPad, San Diego,Calif.). EC₅₀ values (mean±standard error) represent the concentrationat which 50 of the maximal effect is achieved. Relative efficacy (%) wasdetermined by comparison with the effect produced by a maximal effectiveconcentration of 2-MeSADP in the same experiment.

[0164] Antagonist IC₅₀ values (mean±standard error) represent theconcentration needed to inhibit by 50% the effect elicited by 10 nM2-MeSADP. The percent of maximal inhibition is equal to 100 minus theresidual fraction of stimulation at the highest antagonistconcentration.

[0165] All concentration-effect curves were repeated in at least threeseparate experiments carried out with different membrane preparationsusing duplicate or triplicate assays. TABLE 3 Stimulation of PLC atturkey erythrocyte P2Y₁ receptors (agonist effect) and the inhibition ofPLC stimulation elicited by 10 nM 2-MeSADP (antagonist effect), for atleast two separate determinations. Agonist Antagonist Effect, Effect,Com- % of maximal EC₅₀, % of maximal IC₅₀, μM^(b) pound increase^(a)μM^(a) inhibition^(b) (n) 1a^(c,a) NE 99 ± 1 0.331 ± 0.059 (MRS (5)2179) 1b^(e) NE 95 ± 1 0.206 ± 0.053 1c^(e) 4 d 96 ± 2 1.85 ± 0.741d^(e) 6 ± 2 d 94 ± 2 0.362 ± 0.119 4a 95 ± 5  0.155 ± NE 0.021 4b NE100 0.157 ± 0.060 4c NE 100 0.0516 ± 0.0008 5 41 ± 13 13.3 34% at 100 μMsmall decrease

[0166] TABLE 4 Synthetic data for nucleotide derivatives, includingstructural verification using high resolution mass spectroscopy andpurity verification using HPLC. FAB (M-H⁺) HPLC (rt; min)^(a) Method, NoFormula Calcd Found System A System B Yield (%)^(b) 2 C₁₀H₁₅O₉N₅P₂410.0267 410.0269 3.53 10.72 B, 21.7 3b C₁₂H₁₉O₈N₅P₂ 422.0631 422.06643.41 8.21 B, 8.0  4a C₁₂H₁₇O₈N₅P₂ 420.0474 420.0482 3.92 7.30 A, 5.5  4bC₁₃H₁₉O₈N₅P₂ 434.0631 434.0622 5.91 7.83 B, 8.3  4c C₁₃H₁₈O₈N₅P₂Cl468.0241 468.0239 8.05 8.54 B, 2.3  5 C₁₂H₁₇O₈N₅P₂ 420.0474 420.04814.02 6.84 A, 7.5  6 C₁₁H₁₆O₈N₅P₂Cl 442.0084 442.0070 6.67 6.82 A, 24.37b C₁₂H₂₀O₁₂N₅P₃ 518.0237 518.0243 4.98 12.74 A, 1.8  7c C₁₂H₁₉O₉N₅P₂438.0580 438.0580 4.63 9.36 B, 50.1 7d C₁₂H₁₈O₉N₅P₂Cl 472.0201 472.01905.67 9.97 B, 31.3 8a C₁₂H₂₀O₈N₆P₂ 437.0740 437.0721 2.37 8.78 8.0 8bC₁₂H₂₁O₁₁N₆P₃ 517.0403 517.0404 2.42 9.23 7.2 8c C₁₂H₂₂O₁₄N₆P₄ 597.0066597.0053 2.96 10.02 4.0

[0167] Abbreviations

[0168] AIBN, 2,2′-azobisisobutyronitrile;

[0169] ATP, adenosine 5′-triphosphate;

[0170] DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene;

[0171] DCTIDS, 1,3-dichlorotetraisopropyl-1,1,3,3,-disiloxane;

[0172] DEAD, diethylazadicarboxylate;

[0173] DEAE, diethylaminoethyl;

[0174] DMAP, 4-dimethylaninopyridine;

[0175] DMF, dimethylformamide;

[0176] DMSO, dimethylsulfoxide;

[0177] FAB, fast atom bombardment (mass spectroscopy);

[0178] HPLC, high pressure liquid chromatography;

[0179] MS, mass spectroscopy;

[0180] HRMS, high resolution mass spectroscopy;

[0181] LDA, lithium diisopropylamide;

[0182] 2-MeSADP, 2-methylthioadenosine-5′-diphosphate;

[0183] TBAP, tetrabutylammonium phosphate;

[0184] TEAA, triethylammoniun acetate;

[0185] THF, tetrahydrofuran;

[0186] All of the references cited herein, including patents, patentapplications, and publications, are hereby incorporated in theirentireties by reference.

[0187] While this invention has been described with an emphasis uponpreferred embodiments, it will be obvious to those of ordinary skill inthe art that variations of the preferred embodiments may be used andthat it is intended that the invention may be practiced otherwise thanas specifically described herein. Accordingly, this invention includesall modifications encompassed within the spirit and scope of theinvention as defined by the following claims.

What is claimed is:
 1. A compound having the formula A-M, wherein A is achemically modified adenine or uracil and M is a constrained cycloalkylgroup, said adenine or uracil is bonded to said constrained cycloalkylgroup, and said compound binds a receptor; or a salt of said compound.2. The compound of claim 1, wherein said receptor is a P1 or P2receptor.
 3. The compound of claim 2, wherein said P1 receptor isselected from the group consisting of A₁,A_(2A), and A₃.
 4. The compoundof claim 2, wherein said P2 receptor is selected from the groupconsisting of P2Y and P2X.
 5. The compound of claim 1, wherein saidconstrained cycloalkyl group includes a cyclopentyl group.
 6. Thecompound of claim 3, wherein said constrained cyclopentyl group is acyclopentyl ring derivatized with a fused cyclopropane bridge.
 7. Thecompound of claim 1, wherein said constrained cycloalkyl group isconstrained in the N-conformation.
 8. The compound of claim 1, whereinsaid constrained cycloalkyl group is constrained in the S-conformation.9. A compound selected from the group consisting of

wherein R₁ is hydrogen, alkyl, cycloalkyl, alkoxy, cycloalkoxy, aryl,arylalkyl, acyl, sulfonyl, arylsulfonyl, thiazolyl or bicyclic alkyl; R₂is hydrogen, halo, alkyl, aryl, arylamino, aryloxide, alkynyl, alkenyl,thiol, cyano, or; R₃, R₄, and R₅, are each independently hydrogen,hydroxyl, alkoxy, alkyl, alkenyl, alkynyl, aryl, acyl, alkylamino,arylamino, phosphoryl, diphosphoryl, triphosphoryl, phosphonyl, boronyl,thiophosphoryl, thiodiphosphoryl, thiotriphosphoryl or vanadyl, and canbe the same or different; R₆ is hydrogen, alkyl, alkenyl, alkynyl,heteroaryl or aminoalkyl; R₇ is methylene, dihalomethyl, carbonyl,sulfoxide; and at least one of R₁, R₂, and R₆ is other than hydrogen; R₈is carbon or nitrogen; or a salt of said compound.
 10. The compound ofclaim 9, wherein R₁ is alkyl, cycloalkyl, alkoxy, aryl, arylalkyl,bicycloalkyl, or sulfonyl.
 11. The compound of claim 9, wherein R₁ ismethyl, cyclopentyl, cyclohexyl, phenyl, R-phenylisopropyl, benzyl, orphenylethyl; R₂ is chloro; and R₆ is C₁-C₆ alkylamino, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl.
 12. The compound of claim 9 or 10, whereinR₁ is further substituted with a member selected from the groupconsisting of hydroxyl, halo, sulfonyl, amino, cyano, alkoxyl, alkyl,alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, sulfonamido, carboxyl,and carboxamido.
 13. The compound of claim 9, wherein R₁ is methyl groupand R₂ is chloro, alkyithio, or arylalkylthio.
 14. The compound of claim9, wherein R₆ is methyl and R₂ is chloro, alkylthio, arylalkylthio orhydrogen.
 15. The compound of claim 9, wherein R₆ is halo and R₂ is achloro, alkylthio, arylalkylthio or hydrogen.
 16. The compound of claim9, wherein R₂ is chloro.
 17. The compound of claim 9, wherein R₁ ismethyl and R₂ is chloro and R₃ is hydrogen.
 18. The compound of claim 9,wherein the compound has the formula

wherein R₁ is iodobenzyl, or cyclopentyl and R₂ is hydrogen or chloro.19. The compound of claim 9, wherein the compound has the formula


20. The compound of claim 9, wherein the compound has the formula


21. A compound selected from the group consisting of:

wherein R₁, R₂, R₉ is hydrogen, alkyl, alkenyl, alkynyl, aminoalkyl andR₃, R₄, and R₅, are each independently hydrogen, hydroxyl, alkoxy,alkyl, alkenyl, alkynyl, aryl, acyl, alkylamino, arylamino, phosphoryl,phosphonyl, boronyl, or vanadyl, and can be the same or different; R₆and R₇ are each independently sulfur or oxygen; and R₁₀ is methylene,dihalomethyl, carbonyl, sulfoxide; or a salt of said compound.
 22. Thecompound of claim 21, wherein R₁ is methyl.
 23. A compound comprising amethanocarbocyclic analog of a chemically modified adenosine or uradinewherein said compound is a P2 receptor ligand; or a salt of saidcompound.
 24. The compound of claim 23, wherein the compound is a P2receptor agonist.
 25. The compound of claim 23, wherein the compound isa P2 receptor antagonist.
 26. The compound of claim 22, wherein said P2receptor is selected from the group consisting of P2Y and P2X.
 27. Thecompound of claim 22, wherein said P2 receptor is a P2Y receptor. 28.The compound of claim 22, wherein said P2 receptor is a P2Y1 receptor.29. The compound of claim 22, wherein said P2 receptor is a P2Xreceptor.
 30. A compound comprising a methanocarbocyclic analog of achemically modified adenosine or uradine wherein said compound is a P1receptor ligand; or a salt of said compound.
 31. The compound of claim30, wherein the compound is a P1 receptor agonist.
 32. The compound ofclaim 30, wherein the compound is a P1 receptor antagonist.
 33. Thecompound of claim 30, wherein said P1 receptor is selected from thegroup consisting of A₁,A_(2A), and A₃.
 34. The compound of claim 30,wherein said P1 receptor is A₁ receptor.
 35. The compound of claim 30,wherein said P1 receptor is A₃ receptor.
 36. A method of treating orpreventing in a mammal a disease, state, or condition that responds toan adenosine, ATP, or UTP receptor agonist or antagonist comprisingadministering to the mammal a compound of any of any of claims 1-36. 37.A pharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of any of claims 1-36.
 33. The use of a compoundof any of claims 1-36 as a medicament.
 34. The use of a methanocarbaanalog in the manufacture of a medicament for the treatment orprevention in a mammal a disease state, or condition that responds to anadenosine, ATP, UTP receptor agonist or antagonist.
 35. A method for thetreatment of airway diseases, cancer, cardiac arrhythmia, cardiacischemia, epilepsy, Huntington's Disease, immunodeficient disorders,inflammatory disorders, neonatal hypoxia, neurodegenerative, pain,Parkinson's Disease, renal failure, schizophrenia, sleep disorders,stroke, thrombosis, urinary incontinence, diabetes, psoriasis, septicshock, brain trauma, glaucoma, or congestive heart failure inindividuals in need of such treatment comprising contacting an effectivequantity of a compound of any of claims 1-36.